Therapeutic antibodies generated using animal immunizations in vivo or by recombinant antibody display technology in vitro have been successful in the clinic and as such have validated these technologies as effective drug discovery techniques. While it is typically expected that monoclonal antibodies derived from animal immunizations are of sufficiently high affinity to achieve therapeutic efficacy, developing therapeutic antibodies from animal immunizations requires either humanization of the non-human antibodies or access to transgenic animals expressing human antibodies. Direct selection of fully human antibodies from pre-established antibody libraries by in vitro display technologies (phage, bacterial, yeast, mammalian cell, and ribosomal displays) (Chao, G. et al. (2006) Nat. Protoc. 1:755-68; Gai, S. A. and Wittrup, K. D. (2007) Curr. Opin. Struct. Biol. 17:467-73; Griffiths, A. D. et al. (1994) EMBO J. 13:3245-60; He, M. and Khan, F. (2005) Expert Rev. Proteomics 2:421-30; Hoogenboom, H. R. (2002) Methods Mol. Biol. 178:1-37; Hoogenboom, H. R. (2005) Nat. Biotechnol. 23:1105-16) offers a valuable parallel approach and may present the best alternative in cases where the target antigen fails to elicit a productive immune response in vivo.
Human antibody libraries have been engineered to display full-length antibody or various antibody fragments such as Fab, scFv, and dAb. The libraries are generally constructed by either capturing and transferring antibody diversities from B cell repertoires into the library with or without additional introduction of synthetic diversity (Hoet, R. M. et al. (2005) Nat. Biotechnol. 23:344-8), or by synthetically randomizing CDR residues in limited human antibody frameworks (Rothe, C. et al. (2008) J. Mol. Biol. 376:1182-200). Because the antibody heavy and light chains are separately amplified and reformatted into the library display format, new pairings between VH and VL are formed during this process. While this VH-VL shuffling allows the creation of novel antigen binding sites, it also very significantly increases the theoretical library diversity. Several strategies have been developed to engineer better and more productive antibody libraries by reducing the theoretical library diversity and the actual library size required to effectively sample the library. These strategies include intelligently designing synthetic or semi-synthetic libraries (Rothe, C. et al. (2008) J. Mol. Biol. 376:1182-200) and immune (Hoet, R. M. et al. (2005) Nat. Biotechnol. 23:344-8) or pseudoimmune (Lee, H. W. et al. (2006) Biochem. Biophys. Res. Commun. 346:896-903) libraries generated from less diverse but potentially more reactive B cell repertoires. Although these approaches may be effective in significantly reducing the theoretical library diversity by several or even many logs, the need for large library sizes and methods to generate them will always complement library designs in order to increase the chances of identifying antibody hits regardless of their scarcity.
The availability of means for the production of nucleic acid libraries and recombinant products produced thereby, such as pharmaceutical proteins, in eukaryotic systems such as yeast, provides significant advantages relative to the use of prokaryotic systems such as E. coli. Yeast can generally be grown to higher cell densities than bacteria and are readily adaptable to continuous fermentation processing. However, the development of yeast species as host/vector systems for the production of recombinant products and libraries is severely hampered by the lack of knowledge about transformation conditions and suitable means for stably introducing foreign nucleic acids into the yeast host cell.
Among the various electrical and biological parameters that facilitate electrotransformation of cells is the adsorption of DNA to the cell surface. Alternating electric fields of low intensity also promote DNA transfer into E. coli bacteria, presumably by the electrical stimulation of DNA permeases. Evidence for the dominant electrodiffusive or electrophoretic effect on electroporative gene transfer of polyelectrolyte DNA has accumulated. Electroosmotic effects and membrane invagination facilitated by electroporation have also been reported.
The application of an electrical field across a yeast cell membrane results in the creation of transient pores that are critical to the electroporation process. An electroporator signal generator provides the voltage (in kV) that travels across the gap (in cm) between the electrodes. This potential difference defines what is called the electric field strength where E equals kV/cm. Each cell has its own critical field strength for optimum electroporation. This is due to cell size, membrane make-up and individual characteristics of the cell wall itself. For example, mammalian cells typically require between 0.5 and 5.0 kV/cm before cell death and/or electroporation occurs. Generally, the required field strength varies inversely with the size of the cell.
Methods of Transformation
1. Transformation by Electroporation    Becker et al. (Methods in Enzymology 194: 182-187 (1991)) disclose methods of transformation of the yeast Saccharomyces cerevisiae (S. cerevisiae). Becker also discloses spheroplast transformation.    Faber et al. (Curr. Genet. 25: 305-310 (1994)) disclose methods for transformation of the methylotropic yeast Hansenula polymorpha. Faber also applied the method to Pichia methanolica.     Helmuth et al. (Analytical Biochem. 293:149-152 (2001)) disclose increased yeast transformation efficiency by combining both LiAc and DTT pretreatments.    Kasutske et al. (Yeast 8: 691-697 (1992)) disclose electropulse transformation of intact Candida maltosa cells by different homologous vector plasmids.    Meilhoc et al. (Bio/Technology 8: 223-227 (1990)) disclose a transformation system using intact S. cerevisiae yeast cells and electric field pulses.    Neumann et al. (1996) Biophys. J. (1996) 71:868-877 disclose kinetics of yeast cell transformation by electroporation and calcium-mediated DNA adsorption.    Piredda et al. (Yeast 10: 1601-1612 (1994)) disclose a transformation system for the yeast Yamadazyma (Pichia) ohmeri.    Scorer et al. (Bio/Technology 12: 181-184 (1994)) disclose P. pastoris vectors allowing for rapid G418 selection of rare high copy number transformants for expression in Pichia pastoris using both electroporation and spheroplast transformation systems.    Sherman et al. (Laboratory Course Manual for Methods in Yeast Genetics, pages 91 102, Cold Spring Harbor Laboratory (1986)) disclose transformation of yeast mutants LEU2 and HIS3.    Suga and Hatakeyama (Curr. Genet. 43:206-211 (2003)) disclose a freezing method to generate competent cells prior to electroporation with addition of calcium.    Thompson et al. (Yeast 14:565-571 (1998)) disclose the preparation of yeast cells such as S. cerevisiae and Candida albicans for transformation by electroporation.    Yang et al. (Applied and Environmental Microbiology 60(12): 4245-4254 (1994)) disclose electroporation of Pichia stipitis based on its URA3 gene and a homologous autonomous replication sequence ARS2.    U.S. Pat. No. 5,716,808 to Raymond discloses methods for preparing Pichia methanolica cells containing foreign DNA constructs using electroporation and methods for producing foreign peptides in Pichia methanolica cells.    U.S. Pat. No. 7,009,045 to Abbas discloses the transformation of the flavinogenic yeasts, Pichia guilliermondii and Candida famata, by electroporaiion and by spheroplast transformation.
2. Transformation by Spheroplast Formation    Becker et al. (Methods in Enzymology 194: 182-187 (1991)) disclose methods of transformation of the yeast S. cerevisiae as well as spheroplast transformation.    Scorer et al. (Bio/Technology 12: 181-184 (1994)) disclose P. pastoris vectors allowing for G418 selection of rare high copy number transformants for expression in Pichia pastoris using both electroporation and spheroplast transformation systems.    U.S. Pat. No. 4,808,537 to Stroman et al. discloses a method for isolating and cloning a methanol inducible gene from Pichia pastoris and the regulatory regions useful for the methanol regulation expression of heterologous genes using spheroplast transformation.    U.S. Pat. No. 4,837,148 to Cregg et al. disclose autonomous replication sequences that are capable of maintaining plasmids as extra-chromosomal elements in host strains of Pichia. The patent further discloses constructs including the DNA sequences as well as transformed organisms produced by spheroplast formation and provides processes for producing the DNA sequences and constructs of the invention, as well as methods for isolating the sequences from any source.    U.S. Pat. No. 4,855,231 to Stroman et al. discloses DNA sequences that are responsive to the presence of methanol, catabolite non-repressing carbon sources and carbon source starvation. The '231 patent demonstrates spheroplast transformation of Pichia pastoris.     U.S. Pat. No. 4,879,231 to Stroman et al. discloses a spheroplast transformation method for the yeast such as Pichia pastoris.     U.S. Pat. No. 4,882,279 to Cregg et al. discloses a spheroplast transformation technique for yeasts of the genus Pichia, for example, Pichia pastoris.     U.S. Pat. No. 5,135,868 to Cregg relates to a method for the site specific genomic modification of yeasts of the genus Pichia. The '868 patent uses a spheroplast transformation method.    U.S. Pat. No. 5,268,273 to Buckholz relates to a method of spheroplast transformation of Pichia pastoris.     U.S. Pat. No. 5,736,383 to Raymond relates to a method of transformation of yeast strains of the genus Pichia, particularly Pichia methanolica. The '383 patent further relates to a method of spheroplast transformation of yeasts of the genus Pichia as well as a method of transformation by electroporation.
3. Other Transformation Systems    Kunze et al. (Current Genetics 9(3): 205-209 (1985)) disclose a method of transformation of S. cerevisiae, Candida maltosa and Pichia guilliermondii G266 with the plasmid pYe(ARG4)-411, which contains the S. cerevisiae ARG4 gene inserted into pBR322. Kunze used CaCl2 in the method of transformation.    Kunze et al. (J. Basic Microbiol. 25(2): 141-144 (1985)) disclose a method of transformation of the industrially important yeasts Candida maltosa and Pichia guilliermondii G266 using CaCl2.    Kunze et al. (Acta Biotechnol. 6(1): 28 (1986)) disclose transformations of the industrially important yeasts Candida maltosa and Pichia guilliermondii.    Neistat et al. (Mol. Ge. Mikrobiol. Virusol. 12: 19-23 (1986))(Abstract only) disclose transformation of Hansenula polymorpha, Pichia guilliermondii, Williopsis saturnus yeast by a plasmid carrying the ADE2 gene of S. cerevisiae. The method of transformation is not disclosed.    U.S. Pat. No. 4,929,555 to Cregg et al. discloses a method for making whole cells of methylotrophic species of genus Pichia competent for transformation by DNA and a method for transforming with DNA whole cells of such species, particularly Pichia pastoris.     U.S. Pat. No. 5,231,007 to Heefner et al. disclose a method of generating and isolating highly flavinogenic strains of Candida famata which produce riboflavin yields of around 7.0 to 7.5 grams per liter per 6 days. The method includes a combination of iterative mutagenizing steps and protoplast fusion steps performed on the parent strain and the descendent strains that are selected following each step according to a screening protocol.
4. Vectors, ARS Elements and Gene Libraries    Clyne, R. K. et al. (EMBO J. 14(24): 6348-6357 (1995)) relates to a fine structure analysis of ARS1, an ARS element of the fission yeast Schizosaccharomyces pombe. Characterization of a series of nested deletion mutations indicated that the minimal fragment of DNA encompassing ARS1 is large since no fragment under 650 bp retained significant ARS activity.    Liauta-Teglivets, O. et al. (Yeast 11(10): 945-952 (1995)) disclose the cloning of the structural gene of GTP-cyclohydrolase involved in riboflavin biosynthesis from a Pichia guilliermondii genomic library.    Cannon, R. D. et al. (Mol. Gen. Genet. 221(2): 210-218 (1990)) disclose isolation and nucleotide sequence of an autonomously replicating sequence (ARS) element functional in Candida albicans and S. cerevisiae.     Takagi, M. et al. (J. Bacteriol. 167(2): 551-555 (1986)) disclose construction of a host-vector system in Candida maltosa by using an ARS site isolated from its genome.    Pla, J. et al. (Gene 165(1): 115-120 (1995)) relates to ARS2 and ARS3 Candida albicans DNA fragments with autonomous replicating activity shown to promote non-integrative genetic transformation of both Candida albicans and S. cerevisiae.     U.S. Pat. No. 5,212,087 to Fournier et al. discloses ARS sequences that are efficacious in Yarrowia lipolytica as well as plasmids carrying these sequences.    U.S. Pat. No. 5,665,600 to Hagenson et al. discloses Pichia pastoris linear plasmids and DNA fragments thereof which contain ARS sequences. The '600 patent used the spheroplast transformation system as described in Cregg et al in U.S. Pat. No. 4,929,555.    U.S. Pat. No. 4,837,148 to Cregg et al. discloses autonomous replication sequences that are capable of maintaining plasmids as extrachromosomal elements in host strains of Pichia. The '148 patent further relates to constructs including the DNA sequences as well as transformed organisms therewith. The patent additionally provides processes for producing the DNA sequences and constructs of the invention, as well as methods for isolating the sequences from any source.    Chao et al. (Nature Protocols, 1 (2):755-768 (2006)) discloses a protocol for transforming yeast cells by electroporation and achieving a maximal 5×107 library size with 5 μg insert and 1 μg vector DNA.
The above methods and disclosures while achieving increasingly higher transformation efficiency are still laborious and take significant time and repetitive efforts to accumulate multiple small libraries in the 106 to 107 size ranges to a larger and combined library size in the 108 to 109 size range.
Yeast libraries have not achieved the size or efficiency that has been achieved by phage libraries. As reviewed by Hoogenboom in 2005 (Nature Biotech., 23(9):1105-1116), a typical maximal phage library size for is 1010 to 1011, whereas a typical yeast library is 107 in size (significantly smaller than those achieved by other display technologies (Hoogenboom, H. R. (2002) Methods Mol. Biol. 178:1-37; Hoogenboom, H. R. (2005) Nat. Biotechnol. 23:1105-16), although library sizes in the 109 range have been previously reported (Hoet, R. M. et al. (2005) Nat. Biotechnol. 23:344-8; Lipovsek, D. et al. (2007) J. Mol. Biol. 368:1024-41; Segal, L. et al. (2007) Bioinformatics 23:1440-9. Feldhaus et al. (Nature Biotech., 21:163-170 (2003)) has shown that a 1.5×109 library can be constructed by laboriously repeating the transformation and then combining transformed libraries. Although recent progress in electroporation protocols (see Chao, Nature Protocols 1(2):755-768 (2006)) has made it possible to achieve a maximal 5×107 yeast library size in a single transformation, we have found that the Chao protocol typically transforms yeast at a significantly lower transformation efficiency. It is still a correct statement that yeast library sizes achieved to date are still significantly below what is routinely achievable by phage display libraries in the 1010 to 1011 size.
Yeast display library selection, using both magnetic bead and fluorescence-activated cell sorting, offers an efficient and sensitive method to enrich specific binders to target antigens, in particular by its compatibility with fluorescence activated cell sorting (FACS). The advantage of this selection power, however, is hampered by the limited size of typical yeast display libraries due to the low transformation efficiency of yeast cells. If the yeast display selection technology could be coupled with large antibody libraries similar to those made for phage display (about 1010 in size), yeast display technology would provide an extremely effective antibody discovery tool.
A need therefore exists for efficient methods for producing protein libraries, e.g., antibody libraries, using yeast.